PROJECT SUMMARY Spermatogenesis is a highly coordinated process that starts with spermatogonial stem cells (SSCs), which self-renew and differentiate into more mature germ cells to sustain male fertility throughout life. Within the seminiferous epithelium, germ cell development depends on a specific environment created by somatic cells, which provide a number of growth and differentiation factors. The interplay between microenvironment and germ cells is crucial for sperm production, as dysregulation of this process will lead to sterility. Our work focuses on the function and regulation of the transcription factor RBPJ in Sertoli cells. RBPJ is the main mediator of NOTCH signaling, and belongs to a repressor/activator complex at the promoter of target genes. Depending on the cellular context, RBPJ indirectly represses or allows the expression of genes such as Gdnf and Cyp26b1, which are critical for the maintenance of undifferentiated spermatogonia. Through a yeast-2-hybrid assay, we have discovered a novel RBPJ-interacting protein in Sertoli cells, called BHC80. BHC80 is a histone reader that presumably stabilizes KDM1A (a H3K4me2 demethylase) at the repressor complex. BHC80 is encoded by the gene Phf21a and is highly expressed in the brain and testis. In humans, mutations (usually microdeletions) in the Phf21a gene cause Potocki-Saffer syndrome, which is associated with severe intellectual disabilities and craniofacial anomalies. In order to understand how BHC80 regulates RBPJ activity, and to study the function of BHC80 and its target genes beyond NOTCH signaling in Sertoli cells, we will establish a mouse Sertoli cell-specific knockout of Phf21a and characterize its testicular phenotype. To produce the floxed Phf21a mouse, we will employ a novel CRISPR/Cas9 technique called DECAI, which is predicted to generate a higher ratio of pups containing the insert. This small research project aligns with NICHD?s Fertility and Infertility?s mission and its high-priority research area of genetic basis of idiopathic male infertility. It is intended for the development of a research methodology, and the established floxed model will be an important resource for research in the areas of reproductive biology, intellectual disabilities and craniofacial anomalies. !